Synthetic peptides are valuable research tools in a variety of biological disciplines. Small peptides are widely used to generate antibodies. Immunologists have found peptides useful for assessing antigenic variation and for studying antigen presentation. Cell biologists employ small peptides to disrupt cell-substrate adhesion and to target proteins to specific cellular compartments. Peptides have long served as model systems in studies on the structure, folding or associations of proteins. Peptides also possess useful therapeutical or pharmacological properties.
Obtaining small peptides using solid phase synthetic techniques is a lengthy, laborious and expensive process. A recent survey snowed the average cost for a 25-residue peptide to be in excess of $2,000. In solid phase synthesis, the peptide must be built one residue at a time, with changes of chemicals between each coupling step. Moreover, repetitive couplings become increasingly necessary as the peptide chain lengthens. Purification of the desired synthetic peptide from among truncated or otherwise aberrant synthetic peptides can also prove very troublesome.
The expression of ubiquitin fusion proteins in E. coli and the subsequent purification are described by Monia et al, J. Biol. Chem. 264, 4093-4103 (Mar. 5, 1989). These carboxyl extension proteins (CEP) are from 52 to 80 amino acids in length and are naturally occurring proteins found in various organisms ranging from yeast to humans.
Ecker et al, J. Biol. Chem. 264, 7715-7719 (May 5, 1989) teach the expression of cloned eucaryotic genes in microorganisms to allow for the isolation of large quantities of naturally occurring protein products which are present in only trace amounts from natural sources. However, Ecker et al. state that expression of these genes in E. coli often leads to gene products which do not fold properly and are not biologically active. Instead, Saccharomyces cerevisiae is stated to be a superior expression host. Protein yield is stated to increase when genes are expressed in yeast by fusion to ubiquitin. The proteins produced in this manner are the subunit of the mammalian stimulating G-protein of the adenylate cyclase complex; a soluble fragment of the T cell receptor protein; and the protease domain of human urokinase.
Butt et al., Proc. Natl. Acad. Sci. 86, 2540-2544 (April 1989) teach an expression system for cloning ubiquitin-fusion proteins using E. coli and state that fusion of ubiquitin by its carboxyl terminal end to the N-terminus of these proteins increases the yield of unstable or poorly expressed proteins such as those referred to by Ecker et al, supra. Butt et al. conclude that ubiquitin fusion technology has the potential for general application in augmenting the yield of cloned gene products in both procaryotes and eucaryotes.
As early as 1986, Bachmair et al., Science 234, 179-186 (1986), suggest that ubiquitin may be helpful in preparing [beta]-galactosidase fusion proteins having any N-terminal amino acid when expressed in both bacteria and yeast. When expressed in Saccharomyces cerevisiae cells, rapid cleavage of ubiquitin from the [beta]-galactosidase occurred with any amino acid except proline. When expressed in E. coli, the fusion proteins were not disassembled.
One disadvantage of expressing ubiquitin fusion proteins in eucaryotic cells is that they contain hydrolases, i.e. peptidases, as natural products which break the junction of the ubiquitin-fusion protein and, in many cases, target the fusion protein for proteolytic degradation after removing ubiquitin. Bachmair et al., supra, do state that joining ubiquitin to the amino-terminus of target proteins, to yield linear ubiquitin fusion products may be feasible by constructing appropriate genes and expressing them in vivo.
In the prior art, the emphasis is on the use of both eucaryotic and procaryotic cell expressions utilizing ubiquitin fusion proteins for the cloning and production of natural intracellular proteins. These natural proteins are often larger than ubiquitin. Wilkinson et al., Arch. Biochem. Biophys. 250, 390-399 (1986), speculate that ubiquitin may undergo conformational changes following attachment to a target protein. It is possible that proteins having residues approaching or exceeding those of ubiquitin dominate, or at least interfere, with the favorable stability properties of the ubiquitin molecule rendering it less effective as a substrate for fusion protein synthesis.